Research ArticleCell Senescence in MyxoidRound Cell Liposarcoma

Christina Karingbjoumlrn Gustafsson1 Anders Staringhlberg1 Katarina Engtroumlm2 Anna Danielsson1

Ingela Turesson3 and Pierre Aringman1

1 Sahlgrenska Cancer Center Department of Pathology Institute of Biomedicine University of Gothenburg Box 42540530 Gothenburg Sweden

2Department of Oncology Institute of Medical Sciences University of Gothenburg Bla straket 2 41345 Gothenburg Sweden3Department of Oncology Institute of Medical Sciences Uppsala University 75185 Uppsala Sweden

Correspondence should be addressed to Pierre Aman pierreamanguse

Received 21 January 2014 Revised 22 May 2014 Accepted 23 May 2014 Published 24 June 2014

Academic Editor Charles Catton

Copyright copy 2014 Christina Kabjorn Gustafsson et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Myxoidround cell liposarcoma (MLSRCLS) is the second most common liposarcoma type and characterized by the fusiononcogenes FUS-DDIT3 or EWSR1-DDIT3 Previous analysis of cell cycle regulatory proteins revealed a prominent expression of G1-cyclins cyclin dependent kinases and their inhibitors but very few cells progressing through the G1S boundary Here we extendthe investigation to proteins involved in cell senescence in an immunohistochemistry based study of 17 MLSRCLS cases Largesubpopulations of tumor cells expressed the RBL2 pocket protein and senescence associated heterochromatin 1120574 and IL8 receptor120573 We conclude that MLSRCLS tissues contain major populations of senescent tumor cells and this may explain the slow growthrate of this tumor type

1 Introduction

Liposarcoma is the most common soft tissue tumor inhumans and myxoidround cell liposarcoma (MLSRCLS) isthe second most common liposarcoma type In a majorityof the cases MLSRCLS develops in large muscles mostoften in the thigh [1] MLSRCLS tissue is composed ofround to oval shaped mesenchymal cells and a variablenumber of lipoblasts set in a myxoid matrix with a finepiped capillary network Most cases of MLS tumors arerelatively slow growing but 10ndash15 show a hyper cellularround cell morphology (RCLS) with less myxoid com-ponent that is associated with an unfavourable prognosis[1]

The fusion oncogenes FUS-DDIT3 or EWSR1-DDIT3 arepresent in almost all cases of MLSRCLS They result fromt(12 16) or t(12 22) chromosome translocation and havecausative roles in development of MLSRCLS [2ndash5] FUS-DDIT3 and EWSR1-DDIT3 encode abnormal transcriptionfactors that deregulate the expression of target genes [6ndash8]

For example we previously showed that FUS-DDIT3 directlyinduces production of IL6 and IL8 by binding to the IL6 andIL8 promoters [9 10]

A recent study reports that most MLSRCLS cases alsocarry TERT promoter mutations suggesting an increasedTERT activity and extended cellular life span [11] The RCLSvariant is also associated with additional mutations mostcommonly in PIK3CA [12]

Previous studies of cell cycle regulating proteins inMLSRCLS revealed a prominent expression of growth pro-moting G1 cyclins and cyclin dependent kinases (CDK)coexpressed with cyclin dependent kinase inhibitors P16 P19and P27 (also known as CDKN2A CDKN2D and CDKN1Bresp) [13] This pattern suggested that the growth promotingcyclinCDK activity was counteracted by CDK inhibitors andthis could explain the low frequency of proliferating cellsidentified by Ki67 and cyclin A staining (typically less than8 and 4 percent resp) [13]

Recent results from investigations of senescent cells sug-gest that they express G1 cyclins CDKs and their inhibitors

Hindawi Publishing CorporationSarcomaVolume 2014 Article ID 208786 7 pageshttpdxdoiorg1011552014208786

2 Sarcoma

Table 1 Cases and immunohistochemistry data

Case Age Site RB11 () HP11205741 () RBL21 () IL8R1 () KI671 () FUSDDIT3rearrangement2

Histologicalclassification

1 42 im Hip 35 50 79 55 nd + MLS2 45 im Thigh 23 66 77 86 lt1 + MLS3 37 im Thigh 73 50 69 69 4 + MLS4 36 im Thigh 60 56 55 86 lt1 + MLS5 80 im Thigh 62 60 93 91 2 + MLS6 34 im Thigh 58 44 91 79 3 + MLS7 56 im Thigh 72 47 76 64 lt1 + MLS8 46 sc Thigh 65 51 85 86 1 + MLS9 73 im Lower leg 75 49 83 83 lt1 nd MLS10 45 other Peritoneal 73 54 80 80 3 nd MLS11 49 sc Thorax met 94 56 93 90 nd + MLS12 33 other Peritoneal 25 76 87 50 lt1 + MLSRCLS13 37 im Leg 9 14 69 62 nd nd MLSRCLS14 38 im Thigh 81 47 68 85 6 + MLSRCLS15 76 sc Back 73 50 82 54 lt1 + MLSRCLS16 46 im Hip 85 45 0 74 8 + RCLS17 19 im Shoulder 77 51 78 79 7 nd RCLS1Percent cells expressing respective protein2FUS-DDIT3 rearrangement status as determined by FISH +Indicates presence of FUS-DDIT3 rearrangements nd not determined due to technical problemsor lack of materialim intramuscular sc subcutaneous nd not determined met metastasis

as well as a typical expression signature of cytokines andtheir receptors [14ndash17] These new data and concepts providea possible explanation for the expression patterns observedin MLSRCLS and prompted a renewed investigation withfocus on senescence markers In the present study wehave employed immunohistochemistry (IHC) and analyseda cohort of 17MLSRCLS cases for expression of proteinsassociated with growth control and senescence The resultssuggest that substantial proportions of MLSRCLS tumorcells are senescent

2 Materials and Methods

21 Tissue Samples The clinicopathological characteristicsof all tumors are presented in Table 1 Formalin-fixedtissues taken at surgery from previously untreated caseswith MLSRCLS were embedded in paraffin using routineprocedures and stored at room temperature Use of tissuesamples for this study was examined and approved by theEthical Board associated with the University of GothenburgAll selectedMLS cases were examined by two clinical pathol-ogists specialized in soft tissue tumors and by the first authoralso a clinical pathologist

22 Cell Cultures Human foreskin derived fibroblasts (pas-sage 8) were cultured in RPMI1640 with 5 fetal calf serumImmunofluorescence studies were made 5 days after a 10Gydose of X-ray radiation MLS cell lines 402-91 1765-92

and 2645-94 and HT1080 clones [4 18 19] were culturedin RPMI1640 with GlutaMAX and 8 fetal bovine serum100UmL penicillin and 100 120583gmL streptomycin All mediaand supplements were obtained from Life Technologies Allcells were maintained at 37∘C with air containing 5 CO

2

23 IL-6 Dependence Assay MLS cell lines 402-91 1765-92 and 2645-94 were seeded to 96 well microtiter plates at2000 cellswell and allowed to settle for 8 hours The culturemedium was replaced with medium supplemented with 3fetal bovine serum with and without 1 unitmL of recombi-nant IL6 and with or without 05120583gmL of monoclonal IL6antibody mAB 206 (RampD systems) Trypan blue excludingcells were counted in an invertedmicroscope after 48 hours ofincubation AnEpstein-Barr virus immortalized lymphoblas-toid cell line was included as positively responding control[20]

24 Immunohistochemistry and ImmunofluorescenceMicroscopy Series of 5 120583m tissue sections were cut fromeach biopsy deparaffinised rehydrated boiled in microwaveoven for 10 minutes for epitope retrievement and stainedwith the antibodies listed in Table 2 Bound antibodies werevisualized using the LSAB secondary antibody streptavidinbiotin peroxidase system (DAKO) Stained sections wereexamined on a light microscope

For immunofluorescence analysis cultures of humanfibroblasts were washed twice with PBS and fixed in 4

Sarcoma 3

Table 2 Antibodies used for immunohistochemistry andimmunofluorescence

Antigen Antibody Dilution RetrievementIL8R120573 SC-23811 1 50 PH6RBL2 SC-53641 1 50 PH9RB1 BD 5544136 1 20 PH9TP53 CalbioOP43A 1 100CCNA NMMS1061-S1 1 15P21 Millipore OP64 1 25P15 SC-56327 1 20HP1120574 Millipore 05-690 1 100 PH6KI67 Dako IR-626 1 1 PH6SC SantaCruz Biotechnology BD Becton Dickinson and Company Calbiocalbiochem NM neomarker epitope retrievement was performed at indi-cated PH by boiling for 5 minutes

paraformaldehyde in PBS After two more washes in PBSthe slides were mounted in an antifade mount containingthe DNA binding dye DAPI (46-diamidino-2-phenylindoledihydrochloride) (Olink Bioscience) and examined on afluorescence microscope Several antibodies were tested foreach antigen and evaluated by IHC staining of tissue sectionscontaining published positive and negative cell populationsThe selected primary antibodies were also tested by westernblot analysis of MLS cell lines and other reference cell linesas described elsewhere [13] Irradiated fibroblasts were usedas a control for the senescence markers used (Table 3)Detection of RB1 expression worked satisfactorily only withthe antibody and protocol detailed in Table 2 Evaluationof IHC stains was made by Christina Kabjorn specialistpathologist and Pierre Aman

25 Flow Cytometric Analysis of Cell Cycle Phase DistributionParaffin-embedded formalin-fixed tissues were dewaxedand single-cell suspensions were prepared and labeledwith propidium iodide (Life Technology catalogue numberP3566) according to the providers protocol and as previouslydescribed [21] Flow cytometric analysis of DNA content wasevaluated using the FACS calibur system (BD Biosciences)TheModFit LT software (Verity SoftwareHouse) was used foranalysis and peak detection to identify distributions of G1 Sand G2 cells

26 FISH Analysis Interphase FISH analysis of FUS-DDIT3and EWSR1-DDIT3 rearrangements [22] was performed onformalin-fixed 1ndash4120583m paraffin tissue sections Three break-apart probes DDIT3 FUSand EWSR1 (Vysis Inc) wereused according to protocols supplied by the manufacturerNuclei were counterstained with 10 120583L 4101584061015840-diamidino-21015840-phenylindole dihydrochloride (DAPI) The sections wereanalyzed and reanalyzed by two independent reviewers Atleast 100 nuclei per section were scored The interpretationof intact fusion and split signals was based on guidelinesrecommended by the manufacturer and from other clinicallaboratories using this method

Table 3 Senescence markers in irradiated fibroblasts

Marker Control IrradiatedIL8R Negative 100++

RBL2 8 12 (mitotic)++ 94 96+

RB1 100++ 30 33+

P53 Negative 100++

CCNA 17 20++ NegativeP21 Negative 100+++

P15 Negative 100+

HP1120574 lt1++ 100+++

Percentage positive cells for respective marker are shown ++++++ indicateweak medium and strong expression respectively Mitotic expression seenonly in mitotic cells Results from two experiments are shown Two valuesare shown when there was variation between the experiments

3 Results

Irradiated fibroblasts were used as a control for the senes-cence markers and antibodies used Our data showed a verystrong expression of all investigated senescence associatedmarkers in irradiated cells compared to control cultured cells(Table 3)

Our Ki67 IHC staining results (Table 1) and previousdata [13] showed that most MLS cases contain very fewKi 67 positive cells typically less than 4 but some RCLStissues contained smaller tissue regionswith up to 8positivecells A previous investigation also showed only few cellsexpressing the S-G2 phase specific cyclin A suggesting thata majority of the cells are arrested in the G1 phase [13]Here our flow cytometry analysis of cell nuclei from twohuman MLSRCLS tissues showed 94 and 96 G1-phasecells respectively (Figure 1) supporting our conclusion thatmost MLSRCLS tumor cells were arrested in the G1 phase ofthe cell cycle

IHC analysis of pocket proteins showed that 9ndash94 of thecells expressed the proliferation associated RB1 and 0ndash93expressed the rest of phase protein RBL2 It is thus obviousthat there are overlaps in expression of the RB1 and RBL2proteins

Our IHC analysis showed only weak signals for the RB1protein in MLS tissues compared to control tissue samplesfrom other tumor types This prompted a further analysis ofRB1 protein expression in MLS cells Western blot analysisshowed a strong expression of a normal sized RB1 protein inall three investigatedMLS derived cell lines (Figure 2) Forcedexpression of FUS-DDIT3 gave no effects on RB1 expressionin the human HT1080 fibrosarcoma cell line (Figure 2)

Between 14 and 76 of the tumor cells stained positivefor heterochromatin protein (HP1120574) (Table 1 and Figure 1)Together with the pocket protein results these data suggestthat a large proportion of the MLSRCLS cells are arrestedin the G1 phase and that the tumors contain a substantialfraction of senescent cells

Cell senescence is associated with a distinct cytokineand cytokine receptor expression signature and FUS-DDIT3is known to induce IL6 and IL8 expression in the tumorcells IHC analysis of MLSRCLS tissues showed that the

4 Sarcoma

2000

1500

1000

500

Relative DNA content

Num

ber o

f nuc

lei

40 12080

G1

G2

(a)

RB1 RBL2

IL8RHP1120574

(b)

Figure 1 (a) Flow cytometry histogram of cell nuclei extracted from paraffin embedded MLSRCLS tumor tissue (Cases 8) showing morethan 95 of the cells in the G1 phase of the cell cycle (red) (b) Immunohistochemistry analysis of RB1 RBL2 HP1120574 and IL8R in MLSRCLStumor tissues Brown staining shows reactivity with the specific antibodies Bars are 100120583m

senescence associated IL8 receptor beta (also known asCXCR2) was expressed in 50ndash91 of the tumor cells (Table 1and Figure 1) We tested MLSRCLS tumor cell lines for invitro growth and survival dependence on IL6 but no effects oftreatment with IL6 or IL6 blocking antibodies were detected(data not shown)

4 Discussion

Our previous analysis of cell cycle regulator expression inMLSRCLS suggested that a majority of the tumor cellswere arrested in the G1 phase of the cell cycle [13] Onlya few percent of the cells escape this arrest as detected

Sarcoma 5

by low numbers of Ki67 and cyclin A positive cells Thisconclusion is supported here by the presentKi67 IHCanalysisof 17MLSRCLS cases and by flow cytometry analysis of twocases

The Ki67 and cytometry analysis was complementedby an IHC based investigation of the pocket proteins RB1and RBL2 (also known as P105 and P130) RB1 and RBL2are important control hubs for cell cycle regulation andproliferation driving transcription factors RB1 is expressedin proliferating cells and is also necessary for induction ofsenescence while RBL2 is expressed in resting and senescentcells Consequently the levels of RB1 decrease and the levelsof RBL2 increase as cells enter a nonproliferative or senescentcell state [23ndash27] Our IHC results for RB1 and RBL2 supportthe previous conclusion that large subpopulations of tumorcells are in a resting state

IHC staining of RB1 in MLSRCLS tissues gave generallyweak signals calling for further analysis Six different RB1specific antibodies and various stainingantigen retrievingconditions were tested (data not shown) Compared to thestaining intensities for RBL2 and HP1120574 the signal was alwaysfound weaker in MLS tumor tissues and RB1 staining wasalso weaker in MLS tissues compared to reference tumortissues of other entities Analysis of MLSRCLS derived celllines showed RB1 protein expression in level with othertumor cell lines and much stronger than the expressionin cultured normal fibroblasts (Figure 2) Comparison ofHT1080 fibrosarcoma cells with and without the FUS-DDIT3showed that the fusion protein has no direct effect on RB1expression (Figure 2) Taken together our data suggest thatMLSRCLS cells are capable of normal expression of RB1Thefading RB1 expression in MLSRCLS may thus result from anormal downregulation in connection with growth cessationof many tumor cells

Some senescent cell types are characterized by nuclearheterochromatin foci that can be visualized by DNA-stainsand the increased expression of heterochromatin protein1 gamma (HP1120574) [28 29] The IHC analysis of HP1120574 inMLSRCLS tissues showed a heterogeneous pattern withlarge numbers of strongly stained cells This suggests thatlarge subpopulations of the tumor cells are senescent withexpanded heterochromatin formation and thus they may bepermanently excluded from further proliferation

FUS-DDIT3 binds the promoter regions of the IL8 andIL6 encoding genes leading to expression of these genes [910] IL6 is reported as an autocrine growth or survival factorin several tumor types [30ndash33] Our results suggested how-ever that IL6 is not a growthsurvival factor for MLSRCLScells More recently IL6 and IL8 were reported to be parts ofa cytokine expression profile (IL6 and IL8) that is typical forsenescent cells [34ndash36] Instead of acting as growth factorsIL6 together with IL8 may thus be part of a senescencemechanism in MLSRCLS Our data showing IL8 receptorexpression in many of the tumor cells is in line with thisinterpretationThe IL8 receptor beta expression also indicatesa possible senescence associated IL8 autocrine activity asthe tumor cells also are producing IL8 [7] A schematicpresentation of the investigated senescence associated factorsin MLSRCLS is shown in Figure 3

Fibr

obla

sts

MLS

402

-91

MLS

176

5-93

MLS

264

5-94

HT1

080

EGFP

HT1

080

FUS-

DD

IT3

ltpRB

Figure 2 Western blot analysis of RB1 expression in culturednormal human fibroblasts MLS cell lines and FUS-DDIT3 or EGFPtransfected HT1080 cells

Senescent cells

IL8R

RB1RBL2

Stem cells

Mar

kers

Progenitors

Lipoblasts

IL6 and IL8

FUS-DDIT3Senescence induction

HP1120574

Figure 3 Schematic presentation of tumor populations and factorsinvolved in senescence A small population of proliferating cellsarises from a hypothetical tumor stem cell population Most ofthese cells enter senescence and a few percent differentiates intolipoblasts RB1 and RBL2 proteins are central factors involvedin growth regulation and entrymaintenance of cell senescenceRB1 is expressed in proliferating cells and is also necessary fordifferentiation and senescence RBL2 is expressed in resting andsenescent cells Increased HP1120574 expression is typical for senescentcells IL6 and IL8 are produced by the tumor cells and IL8 may bindto the IL8120573 receptor expressed by senescent tumor cells

Our hypothesis that major subpopulations of MLSRCLScells are senescent may seem contradictory to the recentreport that MLSRCLS tumors carry TERT promoter muta-tions Such mutations may indicate an increased TERTactivity and immortalization of the tumor cells [11] Thelarge numbers of senescent cells in MLSRCLS tumors mayhowever be caused by oncogene induced stress responsesresulting in senescence This hypothesis is supported by ourprevious in vitro experiments with FUS-DDIT3 transfectioninto various cell types Forced FUS-DDIT3 expression causedcell death and senescence in most cell types and only veryfew cells in permissive cell lines maintained proliferativecapacity [18] Surviving FUS-DDIT3 transfected cells alsohad a slower in vivo and in vitro growth rate compared towild-type cells [18] Oncogenic stress induced senescencehas been reported for many oncogenes and is thought toserve as a major barrier against tumor development in vivo[14ndash17] This mechanism may also explain the observed lowgrowth rate and abundance of senescent cells in MLSRCLStumors

6 Sarcoma

Ten to fifteen percent of MLSRCLS cases show roundcell morphology and this is associated with increased growthrate and unfavorable prognosis Inspection of our limitedcohort of cases failed to detect any correlation between theRCLSmorphology and expression data of the analyzedmark-ers A possible association between RCLS morphology andnumber of senescent cells has to be tested in a larger tumormaterial

Irradiated fibroblasts showed a very strong expressionof all investigated senescence associated markers In a clin-ical study we have previously reported that irradiation ofMLSRCLS tumors results in transformation to a lipoma likemorphology with highly differentiated adipocyte like cells[37] Further investigations of irradiated MLSRCLS tumorswill show if this treatment causes maturation of senescentcells into adipocyte like cells Such effects have been reportedin other tumor types [38]

We have investigated 17MLSRCLS tumors for expressionof senescence associated proteins The results suggest thatlarge subpopulations of tumor cells are in a senescent cell statecharacterized by expression of HP1120574 RBL2 and senescenceassociated cytokines and a cytokine receptor The presenceof large numbers of senescent cells may explain the observedslow growth rate of this sarcoma type

Conflict of Interests

None of the authors have any economical or other conflictinginterests with regard to the publication of this paper

Acknowledgments

This work was supported by grants from the Swedish Can-cer Society Swedish Research Council LUAALF grantsVastra Gotaland Assar Gabrielssons Research FoundationJohan Jansson Foundation for Cancer Research SwedishSociety for Medical Research BioCARE National Strate-gic Research Program at University of Gothenburg andWilhelm and Martina Lundgren Foundation for ScientificResearch

References

[1] C D Fletcher K K Unni and F Mertens Tumors of SoftTissue and Bone WHO Classification Pathology and GeneticsIARCPress Lyon France 2000

[2] A Crozat P Aman N Mandahl and D Ron ldquoFusion ofCHOP to a novel RNA-binding protein in human myxoidliposarcomardquo Nature vol 363 no 6430 pp 640ndash644 1993

[3] T H Rabbitts A Forster R Larson and P Nathan ldquoFusionof the dominant negative transcription regulator CHOP witha novel gene FUS by translocation t(1216) in malignant liposar-comardquo Nature Genetics vol 4 no 2 pp 175ndash180 1993

[4] P Aman D Ron N Mandahl et al ldquoRearrangement of thetranscription factor gene CHOP in myxoid liposarcomas witht(1216)(q13p11)rdquo Genes Chromosomes and Cancer vol 5 no 4pp 278ndash285 1992

[5] I Panagopoulos M Hoglund F Mertens N Mandahl FMitelman and P Aman ldquoFusion of the EWS and CHOP genes

in myxoid liposarcomardquo Oncogene vol 12 no 3 pp 489ndash4941996

[6] N Riggi L Cironi P Provero et al ldquoExpression of the FUS-CHOP fusion protein in primary mesenchymal progenitor cellsgives rise to a model of myxoid liposarcomardquo Cancer Researchvol 66 no 14 pp 7016ndash7023 2006

[7] H Zinszner R Albalat and D Ron ldquoA novel effector domainfrom the RNA-binding protein TLS or EWS is required foroncogenic transformation by CHOPrdquo Genes and Developmentvol 8 no 21 pp 2513ndash2526 1994

[8] H Zinszner D Immanuel Y Yin F-X Liang and D Ron ldquoAtopogenic role for the oncogenic N-terminus of TLS nucleolarlocalization when transcription is inhibitedrdquo Oncogene vol 14no 4 pp 451ndash461 1997

[9] M Goransson M K Andersson C Forni et al ldquoThe myxoidliposarcoma FUS-DDIT3 fusion oncoprotein deregulates NF-120581B target genes by interaction with NFKBIZrdquoOncogene vol 28no 2 pp 270ndash278 2009

[10] M Goransson E Elias A Stahlberg A Olofsson C Ander-sson and P Aman ldquoMyxoid liposarcoma FUS-DDIT3 fusiononcogene induces CEBP 120573-mediated interleukin 6 expressionrdquoInternational Journal of Cancer vol 115 no 4 pp 556ndash5602005

[11] P J Killela Z J Reitmana Y Jiao et al ldquoTERT promotermutations occur frequently in gliomas and a subset of tumorsderived from cells with low rates of self-renewalrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 110 no 15 pp 6021ndash6026 2013

[12] J Barretina B S Taylor S Banerji et al ldquoSubtype-specificgenomic alterations define new targets for soft-tissue sarcomatherapyrdquo Nature Genetics vol 42 no 8 pp 715ndash721 2010

[13] A Olofsson H Willen M Goransson et al ldquoAbnormalexpression of cell cycle regulators in FUS-CHOP carryingliposarcomasrdquo International Journal of Oncology vol 25 no 5pp 1349ndash1355 2004

[14] M Braig S Lee C Loddenkemper et al ldquoOncogene-inducedsenescence as an initial barrier in lymphoma developmentrdquoNature vol 436 no 7051 pp 660ndash665 2005

[15] J Bartkova N Rezaei M Liontos et al ldquoOncogene-inducedsenescence is part of the tumorigenesis barrier imposed byDNAdamage checkpointsrdquo Nature vol 444 no 7119 pp 633ndash6372006

[16] M Braig and C A Schmitt ldquoOncogene-induced senescenceputting the brakes on tumor developmentrdquo Cancer Researchvol 66 no 6 pp 2881ndash2884 2006

[17] P Yaswen and J Campisi ldquoOncogene-induced senescencepathways weave an intricate tapestryrdquo Cell vol 128 no 2 pp233ndash234 2007

[18] K Engstrom H Willen C Kabjorn-Gustafsson et al ldquoThemyxoidround cell liposarcoma fusion oncogene FUS-DDIT3and the normal DDIT3 induce a liposarcoma phenotype intransfected human fibrosarcoma cellsrdquo American Journal ofPathology vol 168 no 5 pp 1642ndash1653 2006

[19] S Thelin-Jarnum C Lassen I Panagopoulos N Mandahland P Aman ldquoIdentification of genes differentially expressedin TLS-CHOP carrying myxoid liposarcomasrdquo InternationalJournal of Cancer vol 83 no 1 pp 30ndash33 1999

[20] T Yokoi T Miyawaki A Yachie K Kato Y Kasahara and NTaniguchi ldquoEpstein-Barr virus-immortalized B cells produceIL-6 as an autocrine growth factorrdquo Immunology vol 70 no 1pp 100ndash105 1990

Sarcoma 7

[21] I Kopf C Hanson U Delle I Verbiene and A WeimarckldquoA rapid and simplified technique for analysis of archivalformalin-fixed paraffin-embedded tissue by fluorescence insitu hybridization (FISH)rdquo Anticancer Research vol 16 no 5pp 2533ndash2536 1996

[22] H Yoshida K Nagao H Ito K Yamamoto and S UshigomeldquoChromosomal translocations in human soft tissue sarcomasby interphase fluorescence in situ hybridizationrdquo PathologyInternational vol 47 no 4 pp 222ndash229 1997

[23] H HelmboldW Deppert andW Bohn ldquoRegulation of cellularsenescence by Rb2p130rdquo Oncogene vol 25 no 38 pp 5257ndash5262 2006

[24] F P Fiorentino C E SymondsMMacAluso and A GiordanoldquoSenescence and p130Rbl2 a new beginning to the endrdquo CellResearch vol 19 no 9 pp 1044ndash1051 2009

[25] S Haferkamp S L Tran T M Becker L L Scurr R F Keffordand H Rizos ldquoThe relative contributions of the p53 and pRbpathways in oncogene-induced melanocyte senescencerdquo Agingvol 1 no 6 pp 542ndash556 2009

[26] H Helmbold N Komm W Deppert and W Bohn ldquoRb2p130is the dominating pocket protein in the p53-p21 DNA damageresponse pathway leading to senescencerdquoOncogene vol 28 no39 pp 3456ndash3467 2009

[27] H Helmbold U Galderisi and W Bohn ldquoThe switch frompRbp105 to Rb2p130 inDNAdamage and cellular senescencerdquoJournal of Cellular Physiology vol 227 no 2 pp 508ndash513 2012

[28] P D Adams ldquoRemodeling chromatin for senescencerdquo AgingCell vol 6 no 4 pp 425ndash427 2007

[29] R Zhang W Chen and P D Adams ldquoMolecular dissectionof formation of senescence-associated heterochromatin focirdquoMolecular and Cellular Biology vol 27 no 6 pp 2343ndash23582007

[30] D Giri andM Ittmann ldquoInterleukin-8 is a paracrine inducer offibroblast growth factor 2 a stromal and epithelial growth factorin benign prostatic hyperplasiardquoAmerican Journal of Pathologyvol 159 no 1 pp 139ndash147 2001

[31] DGiriMOzen andM Ittmann ldquoInterleukin-6 is an autocrinegrowth factor in human prostate cancerrdquo American Journal ofPathology vol 159 no 6 pp 2159ndash2165 2001

[32] M M Kawano H Ishikawa N Tsuyama et al ldquoGrowth mech-anism of human myeloma cells by interleukin-6rdquo InternationalJournal of Hematology vol 76 supplement 1 pp 329ndash333 2002

[33] H Nakanishi K Yoshioka S Joyama et al ldquoInterleukin-6soluble interleukin-6 receptor signaling attenuates prolifer-ation and invasion and induces morphological changes of anewly established pleomorphic malignant fibrous histiocytomacell linerdquo American Journal of Pathology vol 165 no 2 pp 471ndash480 2004

[34] J C Acosta A OLoghlen A Banito et al ldquoChemokinesignaling via the CXCR2 receptor reinforces senescencerdquo Cellvol 133 no 6 pp 1006ndash1018 2008

[35] J C Acosta A OLoghlen A Banito S Raguz and J GilldquoControl of senescence by CXCR2 and its ligandsrdquo Cell Cyclevol 7 no 19 pp 2956ndash2959 2008

[36] T Kuilman andD S Peeper ldquoSenescence-messaging secretomeSMS-ing cellular stressrdquoNature Reviews Cancer vol 9 no 2 pp81ndash94 2009

[37] K Engstrom P Bergh C-G Cederlund et al ldquoIrradiation ofmyxoidround cell liposarcoma induces volume reduction andlipoma-like morphologyrdquo Acta Oncologica vol 46 no 6 pp838ndash845 2007

[38] K-H von Wangenheim H-P Peterson and K SchwenkeldquoA major component of radiation action interference withintracellular control of differentiationrdquo International Journal ofRadiation Biology vol 68 no 4 pp 369ndash388 1995

4 Sarcoma

2000

1500

1000

500

Relative DNA content

Num

ber o

f nuc

lei

40 12080

G1

G2

(a)

RB1 RBL2

IL8RHP1120574

(b)

Figure 1 (a) Flow cytometry histogram of cell nuclei extracted from paraffin embedded MLSRCLS tumor tissue (Cases 8) showing morethan 95 of the cells in the G1 phase of the cell cycle (red) (b) Immunohistochemistry analysis of RB1 RBL2 HP1120574 and IL8R in MLSRCLStumor tissues Brown staining shows reactivity with the specific antibodies Bars are 100120583m

senescence associated IL8 receptor beta (also known asCXCR2) was expressed in 50ndash91 of the tumor cells (Table 1and Figure 1) We tested MLSRCLS tumor cell lines for invitro growth and survival dependence on IL6 but no effects oftreatment with IL6 or IL6 blocking antibodies were detected(data not shown)

4 Discussion

Our previous analysis of cell cycle regulator expression inMLSRCLS suggested that a majority of the tumor cellswere arrested in the G1 phase of the cell cycle [13] Onlya few percent of the cells escape this arrest as detected

Sarcoma 5

by low numbers of Ki67 and cyclin A positive cells Thisconclusion is supported here by the presentKi67 IHCanalysisof 17MLSRCLS cases and by flow cytometry analysis of twocases

The Ki67 and cytometry analysis was complementedby an IHC based investigation of the pocket proteins RB1and RBL2 (also known as P105 and P130) RB1 and RBL2are important control hubs for cell cycle regulation andproliferation driving transcription factors RB1 is expressedin proliferating cells and is also necessary for induction ofsenescence while RBL2 is expressed in resting and senescentcells Consequently the levels of RB1 decrease and the levelsof RBL2 increase as cells enter a nonproliferative or senescentcell state [23ndash27] Our IHC results for RB1 and RBL2 supportthe previous conclusion that large subpopulations of tumorcells are in a resting state

IHC staining of RB1 in MLSRCLS tissues gave generallyweak signals calling for further analysis Six different RB1specific antibodies and various stainingantigen retrievingconditions were tested (data not shown) Compared to thestaining intensities for RBL2 and HP1120574 the signal was alwaysfound weaker in MLS tumor tissues and RB1 staining wasalso weaker in MLS tissues compared to reference tumortissues of other entities Analysis of MLSRCLS derived celllines showed RB1 protein expression in level with othertumor cell lines and much stronger than the expressionin cultured normal fibroblasts (Figure 2) Comparison ofHT1080 fibrosarcoma cells with and without the FUS-DDIT3showed that the fusion protein has no direct effect on RB1expression (Figure 2) Taken together our data suggest thatMLSRCLS cells are capable of normal expression of RB1Thefading RB1 expression in MLSRCLS may thus result from anormal downregulation in connection with growth cessationof many tumor cells

Some senescent cell types are characterized by nuclearheterochromatin foci that can be visualized by DNA-stainsand the increased expression of heterochromatin protein1 gamma (HP1120574) [28 29] The IHC analysis of HP1120574 inMLSRCLS tissues showed a heterogeneous pattern withlarge numbers of strongly stained cells This suggests thatlarge subpopulations of the tumor cells are senescent withexpanded heterochromatin formation and thus they may bepermanently excluded from further proliferation

FUS-DDIT3 binds the promoter regions of the IL8 andIL6 encoding genes leading to expression of these genes [910] IL6 is reported as an autocrine growth or survival factorin several tumor types [30ndash33] Our results suggested how-ever that IL6 is not a growthsurvival factor for MLSRCLScells More recently IL6 and IL8 were reported to be parts ofa cytokine expression profile (IL6 and IL8) that is typical forsenescent cells [34ndash36] Instead of acting as growth factorsIL6 together with IL8 may thus be part of a senescencemechanism in MLSRCLS Our data showing IL8 receptorexpression in many of the tumor cells is in line with thisinterpretationThe IL8 receptor beta expression also indicatesa possible senescence associated IL8 autocrine activity asthe tumor cells also are producing IL8 [7] A schematicpresentation of the investigated senescence associated factorsin MLSRCLS is shown in Figure 3

Fibr

obla

sts

MLS

402

-91

MLS

176

5-93

MLS

264

5-94

HT1

080

EGFP

HT1

080

FUS-

DD

IT3

ltpRB

Figure 2 Western blot analysis of RB1 expression in culturednormal human fibroblasts MLS cell lines and FUS-DDIT3 or EGFPtransfected HT1080 cells

Senescent cells

IL8R

RB1RBL2

Stem cells

Mar

kers

Progenitors

Lipoblasts

IL6 and IL8

FUS-DDIT3Senescence induction

HP1120574

Figure 3 Schematic presentation of tumor populations and factorsinvolved in senescence A small population of proliferating cellsarises from a hypothetical tumor stem cell population Most ofthese cells enter senescence and a few percent differentiates intolipoblasts RB1 and RBL2 proteins are central factors involvedin growth regulation and entrymaintenance of cell senescenceRB1 is expressed in proliferating cells and is also necessary fordifferentiation and senescence RBL2 is expressed in resting andsenescent cells Increased HP1120574 expression is typical for senescentcells IL6 and IL8 are produced by the tumor cells and IL8 may bindto the IL8120573 receptor expressed by senescent tumor cells

Our hypothesis that major subpopulations of MLSRCLScells are senescent may seem contradictory to the recentreport that MLSRCLS tumors carry TERT promoter muta-tions Such mutations may indicate an increased TERTactivity and immortalization of the tumor cells [11] Thelarge numbers of senescent cells in MLSRCLS tumors mayhowever be caused by oncogene induced stress responsesresulting in senescence This hypothesis is supported by ourprevious in vitro experiments with FUS-DDIT3 transfectioninto various cell types Forced FUS-DDIT3 expression causedcell death and senescence in most cell types and only veryfew cells in permissive cell lines maintained proliferativecapacity [18] Surviving FUS-DDIT3 transfected cells alsohad a slower in vivo and in vitro growth rate compared towild-type cells [18] Oncogenic stress induced senescencehas been reported for many oncogenes and is thought toserve as a major barrier against tumor development in vivo[14ndash17] This mechanism may also explain the observed lowgrowth rate and abundance of senescent cells in MLSRCLStumors

6 Sarcoma

Ten to fifteen percent of MLSRCLS cases show roundcell morphology and this is associated with increased growthrate and unfavorable prognosis Inspection of our limitedcohort of cases failed to detect any correlation between theRCLSmorphology and expression data of the analyzedmark-ers A possible association between RCLS morphology andnumber of senescent cells has to be tested in a larger tumormaterial

Irradiated fibroblasts showed a very strong expressionof all investigated senescence associated markers In a clin-ical study we have previously reported that irradiation ofMLSRCLS tumors results in transformation to a lipoma likemorphology with highly differentiated adipocyte like cells[37] Further investigations of irradiated MLSRCLS tumorswill show if this treatment causes maturation of senescentcells into adipocyte like cells Such effects have been reportedin other tumor types [38]

We have investigated 17MLSRCLS tumors for expressionof senescence associated proteins The results suggest thatlarge subpopulations of tumor cells are in a senescent cell statecharacterized by expression of HP1120574 RBL2 and senescenceassociated cytokines and a cytokine receptor The presenceof large numbers of senescent cells may explain the observedslow growth rate of this sarcoma type

Conflict of Interests

None of the authors have any economical or other conflictinginterests with regard to the publication of this paper

Acknowledgments

This work was supported by grants from the Swedish Can-cer Society Swedish Research Council LUAALF grantsVastra Gotaland Assar Gabrielssons Research FoundationJohan Jansson Foundation for Cancer Research SwedishSociety for Medical Research BioCARE National Strate-gic Research Program at University of Gothenburg andWilhelm and Martina Lundgren Foundation for ScientificResearch

References

[1] C D Fletcher K K Unni and F Mertens Tumors of SoftTissue and Bone WHO Classification Pathology and GeneticsIARCPress Lyon France 2000

[2] A Crozat P Aman N Mandahl and D Ron ldquoFusion ofCHOP to a novel RNA-binding protein in human myxoidliposarcomardquo Nature vol 363 no 6430 pp 640ndash644 1993

[3] T H Rabbitts A Forster R Larson and P Nathan ldquoFusionof the dominant negative transcription regulator CHOP witha novel gene FUS by translocation t(1216) in malignant liposar-comardquo Nature Genetics vol 4 no 2 pp 175ndash180 1993

[4] P Aman D Ron N Mandahl et al ldquoRearrangement of thetranscription factor gene CHOP in myxoid liposarcomas witht(1216)(q13p11)rdquo Genes Chromosomes and Cancer vol 5 no 4pp 278ndash285 1992

[5] I Panagopoulos M Hoglund F Mertens N Mandahl FMitelman and P Aman ldquoFusion of the EWS and CHOP genes

in myxoid liposarcomardquo Oncogene vol 12 no 3 pp 489ndash4941996

[6] N Riggi L Cironi P Provero et al ldquoExpression of the FUS-CHOP fusion protein in primary mesenchymal progenitor cellsgives rise to a model of myxoid liposarcomardquo Cancer Researchvol 66 no 14 pp 7016ndash7023 2006

[7] H Zinszner R Albalat and D Ron ldquoA novel effector domainfrom the RNA-binding protein TLS or EWS is required foroncogenic transformation by CHOPrdquo Genes and Developmentvol 8 no 21 pp 2513ndash2526 1994

[8] H Zinszner D Immanuel Y Yin F-X Liang and D Ron ldquoAtopogenic role for the oncogenic N-terminus of TLS nucleolarlocalization when transcription is inhibitedrdquo Oncogene vol 14no 4 pp 451ndash461 1997

[9] M Goransson M K Andersson C Forni et al ldquoThe myxoidliposarcoma FUS-DDIT3 fusion oncoprotein deregulates NF-120581B target genes by interaction with NFKBIZrdquoOncogene vol 28no 2 pp 270ndash278 2009

[10] M Goransson E Elias A Stahlberg A Olofsson C Ander-sson and P Aman ldquoMyxoid liposarcoma FUS-DDIT3 fusiononcogene induces CEBP 120573-mediated interleukin 6 expressionrdquoInternational Journal of Cancer vol 115 no 4 pp 556ndash5602005

[11] P J Killela Z J Reitmana Y Jiao et al ldquoTERT promotermutations occur frequently in gliomas and a subset of tumorsderived from cells with low rates of self-renewalrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 110 no 15 pp 6021ndash6026 2013

[12] J Barretina B S Taylor S Banerji et al ldquoSubtype-specificgenomic alterations define new targets for soft-tissue sarcomatherapyrdquo Nature Genetics vol 42 no 8 pp 715ndash721 2010

[13] A Olofsson H Willen M Goransson et al ldquoAbnormalexpression of cell cycle regulators in FUS-CHOP carryingliposarcomasrdquo International Journal of Oncology vol 25 no 5pp 1349ndash1355 2004

[14] M Braig S Lee C Loddenkemper et al ldquoOncogene-inducedsenescence as an initial barrier in lymphoma developmentrdquoNature vol 436 no 7051 pp 660ndash665 2005

[15] J Bartkova N Rezaei M Liontos et al ldquoOncogene-inducedsenescence is part of the tumorigenesis barrier imposed byDNAdamage checkpointsrdquo Nature vol 444 no 7119 pp 633ndash6372006

[16] M Braig and C A Schmitt ldquoOncogene-induced senescenceputting the brakes on tumor developmentrdquo Cancer Researchvol 66 no 6 pp 2881ndash2884 2006

[17] P Yaswen and J Campisi ldquoOncogene-induced senescencepathways weave an intricate tapestryrdquo Cell vol 128 no 2 pp233ndash234 2007

[18] K Engstrom H Willen C Kabjorn-Gustafsson et al ldquoThemyxoidround cell liposarcoma fusion oncogene FUS-DDIT3and the normal DDIT3 induce a liposarcoma phenotype intransfected human fibrosarcoma cellsrdquo American Journal ofPathology vol 168 no 5 pp 1642ndash1653 2006

[19] S Thelin-Jarnum C Lassen I Panagopoulos N Mandahland P Aman ldquoIdentification of genes differentially expressedin TLS-CHOP carrying myxoid liposarcomasrdquo InternationalJournal of Cancer vol 83 no 1 pp 30ndash33 1999

[20] T Yokoi T Miyawaki A Yachie K Kato Y Kasahara and NTaniguchi ldquoEpstein-Barr virus-immortalized B cells produceIL-6 as an autocrine growth factorrdquo Immunology vol 70 no 1pp 100ndash105 1990

Sarcoma 7

[21] I Kopf C Hanson U Delle I Verbiene and A WeimarckldquoA rapid and simplified technique for analysis of archivalformalin-fixed paraffin-embedded tissue by fluorescence insitu hybridization (FISH)rdquo Anticancer Research vol 16 no 5pp 2533ndash2536 1996

[22] H Yoshida K Nagao H Ito K Yamamoto and S UshigomeldquoChromosomal translocations in human soft tissue sarcomasby interphase fluorescence in situ hybridizationrdquo PathologyInternational vol 47 no 4 pp 222ndash229 1997

[23] H HelmboldW Deppert andW Bohn ldquoRegulation of cellularsenescence by Rb2p130rdquo Oncogene vol 25 no 38 pp 5257ndash5262 2006

[24] F P Fiorentino C E SymondsMMacAluso and A GiordanoldquoSenescence and p130Rbl2 a new beginning to the endrdquo CellResearch vol 19 no 9 pp 1044ndash1051 2009

[25] S Haferkamp S L Tran T M Becker L L Scurr R F Keffordand H Rizos ldquoThe relative contributions of the p53 and pRbpathways in oncogene-induced melanocyte senescencerdquo Agingvol 1 no 6 pp 542ndash556 2009

[26] H Helmbold N Komm W Deppert and W Bohn ldquoRb2p130is the dominating pocket protein in the p53-p21 DNA damageresponse pathway leading to senescencerdquoOncogene vol 28 no39 pp 3456ndash3467 2009

[27] H Helmbold U Galderisi and W Bohn ldquoThe switch frompRbp105 to Rb2p130 inDNAdamage and cellular senescencerdquoJournal of Cellular Physiology vol 227 no 2 pp 508ndash513 2012

[28] P D Adams ldquoRemodeling chromatin for senescencerdquo AgingCell vol 6 no 4 pp 425ndash427 2007

[29] R Zhang W Chen and P D Adams ldquoMolecular dissectionof formation of senescence-associated heterochromatin focirdquoMolecular and Cellular Biology vol 27 no 6 pp 2343ndash23582007

[30] D Giri andM Ittmann ldquoInterleukin-8 is a paracrine inducer offibroblast growth factor 2 a stromal and epithelial growth factorin benign prostatic hyperplasiardquoAmerican Journal of Pathologyvol 159 no 1 pp 139ndash147 2001

[31] DGiriMOzen andM Ittmann ldquoInterleukin-6 is an autocrinegrowth factor in human prostate cancerrdquo American Journal ofPathology vol 159 no 6 pp 2159ndash2165 2001

[32] M M Kawano H Ishikawa N Tsuyama et al ldquoGrowth mech-anism of human myeloma cells by interleukin-6rdquo InternationalJournal of Hematology vol 76 supplement 1 pp 329ndash333 2002

[33] H Nakanishi K Yoshioka S Joyama et al ldquoInterleukin-6soluble interleukin-6 receptor signaling attenuates prolifer-ation and invasion and induces morphological changes of anewly established pleomorphic malignant fibrous histiocytomacell linerdquo American Journal of Pathology vol 165 no 2 pp 471ndash480 2004

[34] J C Acosta A OLoghlen A Banito et al ldquoChemokinesignaling via the CXCR2 receptor reinforces senescencerdquo Cellvol 133 no 6 pp 1006ndash1018 2008

[35] J C Acosta A OLoghlen A Banito S Raguz and J GilldquoControl of senescence by CXCR2 and its ligandsrdquo Cell Cyclevol 7 no 19 pp 2956ndash2959 2008

[36] T Kuilman andD S Peeper ldquoSenescence-messaging secretomeSMS-ing cellular stressrdquoNature Reviews Cancer vol 9 no 2 pp81ndash94 2009

[37] K Engstrom P Bergh C-G Cederlund et al ldquoIrradiation ofmyxoidround cell liposarcoma induces volume reduction andlipoma-like morphologyrdquo Acta Oncologica vol 46 no 6 pp838ndash845 2007

[38] K-H von Wangenheim H-P Peterson and K SchwenkeldquoA major component of radiation action interference withintracellular control of differentiationrdquo International Journal ofRadiation Biology vol 68 no 4 pp 369ndash388 1995

Sarcoma 5

by low numbers of Ki67 and cyclin A positive cells Thisconclusion is supported here by the presentKi67 IHCanalysisof 17MLSRCLS cases and by flow cytometry analysis of twocases

The Ki67 and cytometry analysis was complementedby an IHC based investigation of the pocket proteins RB1and RBL2 (also known as P105 and P130) RB1 and RBL2are important control hubs for cell cycle regulation andproliferation driving transcription factors RB1 is expressedin proliferating cells and is also necessary for induction ofsenescence while RBL2 is expressed in resting and senescentcells Consequently the levels of RB1 decrease and the levelsof RBL2 increase as cells enter a nonproliferative or senescentcell state [23ndash27] Our IHC results for RB1 and RBL2 supportthe previous conclusion that large subpopulations of tumorcells are in a resting state

IHC staining of RB1 in MLSRCLS tissues gave generallyweak signals calling for further analysis Six different RB1specific antibodies and various stainingantigen retrievingconditions were tested (data not shown) Compared to thestaining intensities for RBL2 and HP1120574 the signal was alwaysfound weaker in MLS tumor tissues and RB1 staining wasalso weaker in MLS tissues compared to reference tumortissues of other entities Analysis of MLSRCLS derived celllines showed RB1 protein expression in level with othertumor cell lines and much stronger than the expressionin cultured normal fibroblasts (Figure 2) Comparison ofHT1080 fibrosarcoma cells with and without the FUS-DDIT3showed that the fusion protein has no direct effect on RB1expression (Figure 2) Taken together our data suggest thatMLSRCLS cells are capable of normal expression of RB1Thefading RB1 expression in MLSRCLS may thus result from anormal downregulation in connection with growth cessationof many tumor cells

Some senescent cell types are characterized by nuclearheterochromatin foci that can be visualized by DNA-stainsand the increased expression of heterochromatin protein1 gamma (HP1120574) [28 29] The IHC analysis of HP1120574 inMLSRCLS tissues showed a heterogeneous pattern withlarge numbers of strongly stained cells This suggests thatlarge subpopulations of the tumor cells are senescent withexpanded heterochromatin formation and thus they may bepermanently excluded from further proliferation

FUS-DDIT3 binds the promoter regions of the IL8 andIL6 encoding genes leading to expression of these genes [910] IL6 is reported as an autocrine growth or survival factorin several tumor types [30ndash33] Our results suggested how-ever that IL6 is not a growthsurvival factor for MLSRCLScells More recently IL6 and IL8 were reported to be parts ofa cytokine expression profile (IL6 and IL8) that is typical forsenescent cells [34ndash36] Instead of acting as growth factorsIL6 together with IL8 may thus be part of a senescencemechanism in MLSRCLS Our data showing IL8 receptorexpression in many of the tumor cells is in line with thisinterpretationThe IL8 receptor beta expression also indicatesa possible senescence associated IL8 autocrine activity asthe tumor cells also are producing IL8 [7] A schematicpresentation of the investigated senescence associated factorsin MLSRCLS is shown in Figure 3

Fibr

obla

sts

MLS

402

-91

MLS

176

5-93

MLS

264

5-94

HT1

080

EGFP

HT1

080

FUS-

DD

IT3

ltpRB

Figure 2 Western blot analysis of RB1 expression in culturednormal human fibroblasts MLS cell lines and FUS-DDIT3 or EGFPtransfected HT1080 cells

Senescent cells

IL8R

RB1RBL2

Stem cells

Mar

kers

Progenitors

Lipoblasts

IL6 and IL8

FUS-DDIT3Senescence induction

HP1120574

Figure 3 Schematic presentation of tumor populations and factorsinvolved in senescence A small population of proliferating cellsarises from a hypothetical tumor stem cell population Most ofthese cells enter senescence and a few percent differentiates intolipoblasts RB1 and RBL2 proteins are central factors involvedin growth regulation and entrymaintenance of cell senescenceRB1 is expressed in proliferating cells and is also necessary fordifferentiation and senescence RBL2 is expressed in resting andsenescent cells Increased HP1120574 expression is typical for senescentcells IL6 and IL8 are produced by the tumor cells and IL8 may bindto the IL8120573 receptor expressed by senescent tumor cells

Our hypothesis that major subpopulations of MLSRCLScells are senescent may seem contradictory to the recentreport that MLSRCLS tumors carry TERT promoter muta-tions Such mutations may indicate an increased TERTactivity and immortalization of the tumor cells [11] Thelarge numbers of senescent cells in MLSRCLS tumors mayhowever be caused by oncogene induced stress responsesresulting in senescence This hypothesis is supported by ourprevious in vitro experiments with FUS-DDIT3 transfectioninto various cell types Forced FUS-DDIT3 expression causedcell death and senescence in most cell types and only veryfew cells in permissive cell lines maintained proliferativecapacity [18] Surviving FUS-DDIT3 transfected cells alsohad a slower in vivo and in vitro growth rate compared towild-type cells [18] Oncogenic stress induced senescencehas been reported for many oncogenes and is thought toserve as a major barrier against tumor development in vivo[14ndash17] This mechanism may also explain the observed lowgrowth rate and abundance of senescent cells in MLSRCLStumors

6 Sarcoma

Ten to fifteen percent of MLSRCLS cases show roundcell morphology and this is associated with increased growthrate and unfavorable prognosis Inspection of our limitedcohort of cases failed to detect any correlation between theRCLSmorphology and expression data of the analyzedmark-ers A possible association between RCLS morphology andnumber of senescent cells has to be tested in a larger tumormaterial

Irradiated fibroblasts showed a very strong expressionof all investigated senescence associated markers In a clin-ical study we have previously reported that irradiation ofMLSRCLS tumors results in transformation to a lipoma likemorphology with highly differentiated adipocyte like cells[37] Further investigations of irradiated MLSRCLS tumorswill show if this treatment causes maturation of senescentcells into adipocyte like cells Such effects have been reportedin other tumor types [38]

We have investigated 17MLSRCLS tumors for expressionof senescence associated proteins The results suggest thatlarge subpopulations of tumor cells are in a senescent cell statecharacterized by expression of HP1120574 RBL2 and senescenceassociated cytokines and a cytokine receptor The presenceof large numbers of senescent cells may explain the observedslow growth rate of this sarcoma type

Conflict of Interests

None of the authors have any economical or other conflictinginterests with regard to the publication of this paper

Acknowledgments

This work was supported by grants from the Swedish Can-cer Society Swedish Research Council LUAALF grantsVastra Gotaland Assar Gabrielssons Research FoundationJohan Jansson Foundation for Cancer Research SwedishSociety for Medical Research BioCARE National Strate-gic Research Program at University of Gothenburg andWilhelm and Martina Lundgren Foundation for ScientificResearch

References

[1] C D Fletcher K K Unni and F Mertens Tumors of SoftTissue and Bone WHO Classification Pathology and GeneticsIARCPress Lyon France 2000

[2] A Crozat P Aman N Mandahl and D Ron ldquoFusion ofCHOP to a novel RNA-binding protein in human myxoidliposarcomardquo Nature vol 363 no 6430 pp 640ndash644 1993

[3] T H Rabbitts A Forster R Larson and P Nathan ldquoFusionof the dominant negative transcription regulator CHOP witha novel gene FUS by translocation t(1216) in malignant liposar-comardquo Nature Genetics vol 4 no 2 pp 175ndash180 1993

[4] P Aman D Ron N Mandahl et al ldquoRearrangement of thetranscription factor gene CHOP in myxoid liposarcomas witht(1216)(q13p11)rdquo Genes Chromosomes and Cancer vol 5 no 4pp 278ndash285 1992

[5] I Panagopoulos M Hoglund F Mertens N Mandahl FMitelman and P Aman ldquoFusion of the EWS and CHOP genes

in myxoid liposarcomardquo Oncogene vol 12 no 3 pp 489ndash4941996

[6] N Riggi L Cironi P Provero et al ldquoExpression of the FUS-CHOP fusion protein in primary mesenchymal progenitor cellsgives rise to a model of myxoid liposarcomardquo Cancer Researchvol 66 no 14 pp 7016ndash7023 2006

[7] H Zinszner R Albalat and D Ron ldquoA novel effector domainfrom the RNA-binding protein TLS or EWS is required foroncogenic transformation by CHOPrdquo Genes and Developmentvol 8 no 21 pp 2513ndash2526 1994

[8] H Zinszner D Immanuel Y Yin F-X Liang and D Ron ldquoAtopogenic role for the oncogenic N-terminus of TLS nucleolarlocalization when transcription is inhibitedrdquo Oncogene vol 14no 4 pp 451ndash461 1997

[9] M Goransson M K Andersson C Forni et al ldquoThe myxoidliposarcoma FUS-DDIT3 fusion oncoprotein deregulates NF-120581B target genes by interaction with NFKBIZrdquoOncogene vol 28no 2 pp 270ndash278 2009

[10] M Goransson E Elias A Stahlberg A Olofsson C Ander-sson and P Aman ldquoMyxoid liposarcoma FUS-DDIT3 fusiononcogene induces CEBP 120573-mediated interleukin 6 expressionrdquoInternational Journal of Cancer vol 115 no 4 pp 556ndash5602005

[11] P J Killela Z J Reitmana Y Jiao et al ldquoTERT promotermutations occur frequently in gliomas and a subset of tumorsderived from cells with low rates of self-renewalrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 110 no 15 pp 6021ndash6026 2013

[12] J Barretina B S Taylor S Banerji et al ldquoSubtype-specificgenomic alterations define new targets for soft-tissue sarcomatherapyrdquo Nature Genetics vol 42 no 8 pp 715ndash721 2010

[13] A Olofsson H Willen M Goransson et al ldquoAbnormalexpression of cell cycle regulators in FUS-CHOP carryingliposarcomasrdquo International Journal of Oncology vol 25 no 5pp 1349ndash1355 2004

[14] M Braig S Lee C Loddenkemper et al ldquoOncogene-inducedsenescence as an initial barrier in lymphoma developmentrdquoNature vol 436 no 7051 pp 660ndash665 2005

[15] J Bartkova N Rezaei M Liontos et al ldquoOncogene-inducedsenescence is part of the tumorigenesis barrier imposed byDNAdamage checkpointsrdquo Nature vol 444 no 7119 pp 633ndash6372006

[16] M Braig and C A Schmitt ldquoOncogene-induced senescenceputting the brakes on tumor developmentrdquo Cancer Researchvol 66 no 6 pp 2881ndash2884 2006

[17] P Yaswen and J Campisi ldquoOncogene-induced senescencepathways weave an intricate tapestryrdquo Cell vol 128 no 2 pp233ndash234 2007

[18] K Engstrom H Willen C Kabjorn-Gustafsson et al ldquoThemyxoidround cell liposarcoma fusion oncogene FUS-DDIT3and the normal DDIT3 induce a liposarcoma phenotype intransfected human fibrosarcoma cellsrdquo American Journal ofPathology vol 168 no 5 pp 1642ndash1653 2006

[19] S Thelin-Jarnum C Lassen I Panagopoulos N Mandahland P Aman ldquoIdentification of genes differentially expressedin TLS-CHOP carrying myxoid liposarcomasrdquo InternationalJournal of Cancer vol 83 no 1 pp 30ndash33 1999

[20] T Yokoi T Miyawaki A Yachie K Kato Y Kasahara and NTaniguchi ldquoEpstein-Barr virus-immortalized B cells produceIL-6 as an autocrine growth factorrdquo Immunology vol 70 no 1pp 100ndash105 1990

Sarcoma 7

[21] I Kopf C Hanson U Delle I Verbiene and A WeimarckldquoA rapid and simplified technique for analysis of archivalformalin-fixed paraffin-embedded tissue by fluorescence insitu hybridization (FISH)rdquo Anticancer Research vol 16 no 5pp 2533ndash2536 1996

[22] H Yoshida K Nagao H Ito K Yamamoto and S UshigomeldquoChromosomal translocations in human soft tissue sarcomasby interphase fluorescence in situ hybridizationrdquo PathologyInternational vol 47 no 4 pp 222ndash229 1997

[23] H HelmboldW Deppert andW Bohn ldquoRegulation of cellularsenescence by Rb2p130rdquo Oncogene vol 25 no 38 pp 5257ndash5262 2006

[24] F P Fiorentino C E SymondsMMacAluso and A GiordanoldquoSenescence and p130Rbl2 a new beginning to the endrdquo CellResearch vol 19 no 9 pp 1044ndash1051 2009

[25] S Haferkamp S L Tran T M Becker L L Scurr R F Keffordand H Rizos ldquoThe relative contributions of the p53 and pRbpathways in oncogene-induced melanocyte senescencerdquo Agingvol 1 no 6 pp 542ndash556 2009

[26] H Helmbold N Komm W Deppert and W Bohn ldquoRb2p130is the dominating pocket protein in the p53-p21 DNA damageresponse pathway leading to senescencerdquoOncogene vol 28 no39 pp 3456ndash3467 2009

[27] H Helmbold U Galderisi and W Bohn ldquoThe switch frompRbp105 to Rb2p130 inDNAdamage and cellular senescencerdquoJournal of Cellular Physiology vol 227 no 2 pp 508ndash513 2012

[28] P D Adams ldquoRemodeling chromatin for senescencerdquo AgingCell vol 6 no 4 pp 425ndash427 2007

[29] R Zhang W Chen and P D Adams ldquoMolecular dissectionof formation of senescence-associated heterochromatin focirdquoMolecular and Cellular Biology vol 27 no 6 pp 2343ndash23582007

[30] D Giri andM Ittmann ldquoInterleukin-8 is a paracrine inducer offibroblast growth factor 2 a stromal and epithelial growth factorin benign prostatic hyperplasiardquoAmerican Journal of Pathologyvol 159 no 1 pp 139ndash147 2001

[31] DGiriMOzen andM Ittmann ldquoInterleukin-6 is an autocrinegrowth factor in human prostate cancerrdquo American Journal ofPathology vol 159 no 6 pp 2159ndash2165 2001

[32] M M Kawano H Ishikawa N Tsuyama et al ldquoGrowth mech-anism of human myeloma cells by interleukin-6rdquo InternationalJournal of Hematology vol 76 supplement 1 pp 329ndash333 2002

[33] H Nakanishi K Yoshioka S Joyama et al ldquoInterleukin-6soluble interleukin-6 receptor signaling attenuates prolifer-ation and invasion and induces morphological changes of anewly established pleomorphic malignant fibrous histiocytomacell linerdquo American Journal of Pathology vol 165 no 2 pp 471ndash480 2004

[34] J C Acosta A OLoghlen A Banito et al ldquoChemokinesignaling via the CXCR2 receptor reinforces senescencerdquo Cellvol 133 no 6 pp 1006ndash1018 2008

[35] J C Acosta A OLoghlen A Banito S Raguz and J GilldquoControl of senescence by CXCR2 and its ligandsrdquo Cell Cyclevol 7 no 19 pp 2956ndash2959 2008

[36] T Kuilman andD S Peeper ldquoSenescence-messaging secretomeSMS-ing cellular stressrdquoNature Reviews Cancer vol 9 no 2 pp81ndash94 2009

[37] K Engstrom P Bergh C-G Cederlund et al ldquoIrradiation ofmyxoidround cell liposarcoma induces volume reduction andlipoma-like morphologyrdquo Acta Oncologica vol 46 no 6 pp838ndash845 2007

[38] K-H von Wangenheim H-P Peterson and K SchwenkeldquoA major component of radiation action interference withintracellular control of differentiationrdquo International Journal ofRadiation Biology vol 68 no 4 pp 369ndash388 1995

6 Sarcoma

Ten to fifteen percent of MLSRCLS cases show roundcell morphology and this is associated with increased growthrate and unfavorable prognosis Inspection of our limitedcohort of cases failed to detect any correlation between theRCLSmorphology and expression data of the analyzedmark-ers A possible association between RCLS morphology andnumber of senescent cells has to be tested in a larger tumormaterial

Irradiated fibroblasts showed a very strong expressionof all investigated senescence associated markers In a clin-ical study we have previously reported that irradiation ofMLSRCLS tumors results in transformation to a lipoma likemorphology with highly differentiated adipocyte like cells[37] Further investigations of irradiated MLSRCLS tumorswill show if this treatment causes maturation of senescentcells into adipocyte like cells Such effects have been reportedin other tumor types [38]

We have investigated 17MLSRCLS tumors for expressionof senescence associated proteins The results suggest thatlarge subpopulations of tumor cells are in a senescent cell statecharacterized by expression of HP1120574 RBL2 and senescenceassociated cytokines and a cytokine receptor The presenceof large numbers of senescent cells may explain the observedslow growth rate of this sarcoma type

Conflict of Interests

None of the authors have any economical or other conflictinginterests with regard to the publication of this paper

Acknowledgments

This work was supported by grants from the Swedish Can-cer Society Swedish Research Council LUAALF grantsVastra Gotaland Assar Gabrielssons Research FoundationJohan Jansson Foundation for Cancer Research SwedishSociety for Medical Research BioCARE National Strate-gic Research Program at University of Gothenburg andWilhelm and Martina Lundgren Foundation for ScientificResearch

References

[1] C D Fletcher K K Unni and F Mertens Tumors of SoftTissue and Bone WHO Classification Pathology and GeneticsIARCPress Lyon France 2000

[2] A Crozat P Aman N Mandahl and D Ron ldquoFusion ofCHOP to a novel RNA-binding protein in human myxoidliposarcomardquo Nature vol 363 no 6430 pp 640ndash644 1993

[3] T H Rabbitts A Forster R Larson and P Nathan ldquoFusionof the dominant negative transcription regulator CHOP witha novel gene FUS by translocation t(1216) in malignant liposar-comardquo Nature Genetics vol 4 no 2 pp 175ndash180 1993

[4] P Aman D Ron N Mandahl et al ldquoRearrangement of thetranscription factor gene CHOP in myxoid liposarcomas witht(1216)(q13p11)rdquo Genes Chromosomes and Cancer vol 5 no 4pp 278ndash285 1992

[5] I Panagopoulos M Hoglund F Mertens N Mandahl FMitelman and P Aman ldquoFusion of the EWS and CHOP genes

in myxoid liposarcomardquo Oncogene vol 12 no 3 pp 489ndash4941996

[6] N Riggi L Cironi P Provero et al ldquoExpression of the FUS-CHOP fusion protein in primary mesenchymal progenitor cellsgives rise to a model of myxoid liposarcomardquo Cancer Researchvol 66 no 14 pp 7016ndash7023 2006

[7] H Zinszner R Albalat and D Ron ldquoA novel effector domainfrom the RNA-binding protein TLS or EWS is required foroncogenic transformation by CHOPrdquo Genes and Developmentvol 8 no 21 pp 2513ndash2526 1994

[8] H Zinszner D Immanuel Y Yin F-X Liang and D Ron ldquoAtopogenic role for the oncogenic N-terminus of TLS nucleolarlocalization when transcription is inhibitedrdquo Oncogene vol 14no 4 pp 451ndash461 1997

[9] M Goransson M K Andersson C Forni et al ldquoThe myxoidliposarcoma FUS-DDIT3 fusion oncoprotein deregulates NF-120581B target genes by interaction with NFKBIZrdquoOncogene vol 28no 2 pp 270ndash278 2009

[10] M Goransson E Elias A Stahlberg A Olofsson C Ander-sson and P Aman ldquoMyxoid liposarcoma FUS-DDIT3 fusiononcogene induces CEBP 120573-mediated interleukin 6 expressionrdquoInternational Journal of Cancer vol 115 no 4 pp 556ndash5602005

[11] P J Killela Z J Reitmana Y Jiao et al ldquoTERT promotermutations occur frequently in gliomas and a subset of tumorsderived from cells with low rates of self-renewalrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 110 no 15 pp 6021ndash6026 2013

[12] J Barretina B S Taylor S Banerji et al ldquoSubtype-specificgenomic alterations define new targets for soft-tissue sarcomatherapyrdquo Nature Genetics vol 42 no 8 pp 715ndash721 2010

[13] A Olofsson H Willen M Goransson et al ldquoAbnormalexpression of cell cycle regulators in FUS-CHOP carryingliposarcomasrdquo International Journal of Oncology vol 25 no 5pp 1349ndash1355 2004

[14] M Braig S Lee C Loddenkemper et al ldquoOncogene-inducedsenescence as an initial barrier in lymphoma developmentrdquoNature vol 436 no 7051 pp 660ndash665 2005

[15] J Bartkova N Rezaei M Liontos et al ldquoOncogene-inducedsenescence is part of the tumorigenesis barrier imposed byDNAdamage checkpointsrdquo Nature vol 444 no 7119 pp 633ndash6372006

[16] M Braig and C A Schmitt ldquoOncogene-induced senescenceputting the brakes on tumor developmentrdquo Cancer Researchvol 66 no 6 pp 2881ndash2884 2006

[17] P Yaswen and J Campisi ldquoOncogene-induced senescencepathways weave an intricate tapestryrdquo Cell vol 128 no 2 pp233ndash234 2007

[18] K Engstrom H Willen C Kabjorn-Gustafsson et al ldquoThemyxoidround cell liposarcoma fusion oncogene FUS-DDIT3and the normal DDIT3 induce a liposarcoma phenotype intransfected human fibrosarcoma cellsrdquo American Journal ofPathology vol 168 no 5 pp 1642ndash1653 2006

[19] S Thelin-Jarnum C Lassen I Panagopoulos N Mandahland P Aman ldquoIdentification of genes differentially expressedin TLS-CHOP carrying myxoid liposarcomasrdquo InternationalJournal of Cancer vol 83 no 1 pp 30ndash33 1999

[20] T Yokoi T Miyawaki A Yachie K Kato Y Kasahara and NTaniguchi ldquoEpstein-Barr virus-immortalized B cells produceIL-6 as an autocrine growth factorrdquo Immunology vol 70 no 1pp 100ndash105 1990

Sarcoma 7

[21] I Kopf C Hanson U Delle I Verbiene and A WeimarckldquoA rapid and simplified technique for analysis of archivalformalin-fixed paraffin-embedded tissue by fluorescence insitu hybridization (FISH)rdquo Anticancer Research vol 16 no 5pp 2533ndash2536 1996

[22] H Yoshida K Nagao H Ito K Yamamoto and S UshigomeldquoChromosomal translocations in human soft tissue sarcomasby interphase fluorescence in situ hybridizationrdquo PathologyInternational vol 47 no 4 pp 222ndash229 1997

[23] H HelmboldW Deppert andW Bohn ldquoRegulation of cellularsenescence by Rb2p130rdquo Oncogene vol 25 no 38 pp 5257ndash5262 2006

[24] F P Fiorentino C E SymondsMMacAluso and A GiordanoldquoSenescence and p130Rbl2 a new beginning to the endrdquo CellResearch vol 19 no 9 pp 1044ndash1051 2009

[25] S Haferkamp S L Tran T M Becker L L Scurr R F Keffordand H Rizos ldquoThe relative contributions of the p53 and pRbpathways in oncogene-induced melanocyte senescencerdquo Agingvol 1 no 6 pp 542ndash556 2009

[26] H Helmbold N Komm W Deppert and W Bohn ldquoRb2p130is the dominating pocket protein in the p53-p21 DNA damageresponse pathway leading to senescencerdquoOncogene vol 28 no39 pp 3456ndash3467 2009

[27] H Helmbold U Galderisi and W Bohn ldquoThe switch frompRbp105 to Rb2p130 inDNAdamage and cellular senescencerdquoJournal of Cellular Physiology vol 227 no 2 pp 508ndash513 2012

[28] P D Adams ldquoRemodeling chromatin for senescencerdquo AgingCell vol 6 no 4 pp 425ndash427 2007

[29] R Zhang W Chen and P D Adams ldquoMolecular dissectionof formation of senescence-associated heterochromatin focirdquoMolecular and Cellular Biology vol 27 no 6 pp 2343ndash23582007

[30] D Giri andM Ittmann ldquoInterleukin-8 is a paracrine inducer offibroblast growth factor 2 a stromal and epithelial growth factorin benign prostatic hyperplasiardquoAmerican Journal of Pathologyvol 159 no 1 pp 139ndash147 2001

[31] DGiriMOzen andM Ittmann ldquoInterleukin-6 is an autocrinegrowth factor in human prostate cancerrdquo American Journal ofPathology vol 159 no 6 pp 2159ndash2165 2001

[32] M M Kawano H Ishikawa N Tsuyama et al ldquoGrowth mech-anism of human myeloma cells by interleukin-6rdquo InternationalJournal of Hematology vol 76 supplement 1 pp 329ndash333 2002

[33] H Nakanishi K Yoshioka S Joyama et al ldquoInterleukin-6soluble interleukin-6 receptor signaling attenuates prolifer-ation and invasion and induces morphological changes of anewly established pleomorphic malignant fibrous histiocytomacell linerdquo American Journal of Pathology vol 165 no 2 pp 471ndash480 2004

[34] J C Acosta A OLoghlen A Banito et al ldquoChemokinesignaling via the CXCR2 receptor reinforces senescencerdquo Cellvol 133 no 6 pp 1006ndash1018 2008

[35] J C Acosta A OLoghlen A Banito S Raguz and J GilldquoControl of senescence by CXCR2 and its ligandsrdquo Cell Cyclevol 7 no 19 pp 2956ndash2959 2008

[36] T Kuilman andD S Peeper ldquoSenescence-messaging secretomeSMS-ing cellular stressrdquoNature Reviews Cancer vol 9 no 2 pp81ndash94 2009

[37] K Engstrom P Bergh C-G Cederlund et al ldquoIrradiation ofmyxoidround cell liposarcoma induces volume reduction andlipoma-like morphologyrdquo Acta Oncologica vol 46 no 6 pp838ndash845 2007

[38] K-H von Wangenheim H-P Peterson and K SchwenkeldquoA major component of radiation action interference withintracellular control of differentiationrdquo International Journal ofRadiation Biology vol 68 no 4 pp 369ndash388 1995

Sarcoma 7

[21] I Kopf C Hanson U Delle I Verbiene and A WeimarckldquoA rapid and simplified technique for analysis of archivalformalin-fixed paraffin-embedded tissue by fluorescence insitu hybridization (FISH)rdquo Anticancer Research vol 16 no 5pp 2533ndash2536 1996

[22] H Yoshida K Nagao H Ito K Yamamoto and S UshigomeldquoChromosomal translocations in human soft tissue sarcomasby interphase fluorescence in situ hybridizationrdquo PathologyInternational vol 47 no 4 pp 222ndash229 1997

[23] H HelmboldW Deppert andW Bohn ldquoRegulation of cellularsenescence by Rb2p130rdquo Oncogene vol 25 no 38 pp 5257ndash5262 2006

[24] F P Fiorentino C E SymondsMMacAluso and A GiordanoldquoSenescence and p130Rbl2 a new beginning to the endrdquo CellResearch vol 19 no 9 pp 1044ndash1051 2009

[25] S Haferkamp S L Tran T M Becker L L Scurr R F Keffordand H Rizos ldquoThe relative contributions of the p53 and pRbpathways in oncogene-induced melanocyte senescencerdquo Agingvol 1 no 6 pp 542ndash556 2009

[26] H Helmbold N Komm W Deppert and W Bohn ldquoRb2p130is the dominating pocket protein in the p53-p21 DNA damageresponse pathway leading to senescencerdquoOncogene vol 28 no39 pp 3456ndash3467 2009

[27] H Helmbold U Galderisi and W Bohn ldquoThe switch frompRbp105 to Rb2p130 inDNAdamage and cellular senescencerdquoJournal of Cellular Physiology vol 227 no 2 pp 508ndash513 2012

[28] P D Adams ldquoRemodeling chromatin for senescencerdquo AgingCell vol 6 no 4 pp 425ndash427 2007

[29] R Zhang W Chen and P D Adams ldquoMolecular dissectionof formation of senescence-associated heterochromatin focirdquoMolecular and Cellular Biology vol 27 no 6 pp 2343ndash23582007

[30] D Giri andM Ittmann ldquoInterleukin-8 is a paracrine inducer offibroblast growth factor 2 a stromal and epithelial growth factorin benign prostatic hyperplasiardquoAmerican Journal of Pathologyvol 159 no 1 pp 139ndash147 2001

[31] DGiriMOzen andM Ittmann ldquoInterleukin-6 is an autocrinegrowth factor in human prostate cancerrdquo American Journal ofPathology vol 159 no 6 pp 2159ndash2165 2001

[32] M M Kawano H Ishikawa N Tsuyama et al ldquoGrowth mech-anism of human myeloma cells by interleukin-6rdquo InternationalJournal of Hematology vol 76 supplement 1 pp 329ndash333 2002

[33] H Nakanishi K Yoshioka S Joyama et al ldquoInterleukin-6soluble interleukin-6 receptor signaling attenuates prolifer-ation and invasion and induces morphological changes of anewly established pleomorphic malignant fibrous histiocytomacell linerdquo American Journal of Pathology vol 165 no 2 pp 471ndash480 2004

[34] J C Acosta A OLoghlen A Banito et al ldquoChemokinesignaling via the CXCR2 receptor reinforces senescencerdquo Cellvol 133 no 6 pp 1006ndash1018 2008

[35] J C Acosta A OLoghlen A Banito S Raguz and J GilldquoControl of senescence by CXCR2 and its ligandsrdquo Cell Cyclevol 7 no 19 pp 2956ndash2959 2008

[36] T Kuilman andD S Peeper ldquoSenescence-messaging secretomeSMS-ing cellular stressrdquoNature Reviews Cancer vol 9 no 2 pp81ndash94 2009

[37] K Engstrom P Bergh C-G Cederlund et al ldquoIrradiation ofmyxoidround cell liposarcoma induces volume reduction andlipoma-like morphologyrdquo Acta Oncologica vol 46 no 6 pp838ndash845 2007

[38] K-H von Wangenheim H-P Peterson and K SchwenkeldquoA major component of radiation action interference withintracellular control of differentiationrdquo International Journal ofRadiation Biology vol 68 no 4 pp 369ndash388 1995